Endothelium-derived nitric oxide (NO) regulates many important aspects of cardiovascular homeostasis and alterations in vascular NO production of as little as two-fold can have major consequences in both normal vascular physiology and vascular pathophysiology. NO has a crucial role in regulation of the state of vasodilatation of blood vessels and hence in regulation of blood pressure. NO released from the endothelium also modulates other processes as well including platelet aggregation, platelet and leukocyte adhesion to the endothelium, vascular smooth muscle cell proliferation, endothelial cell apoptosis, angiogenesis, and vascular leakage involved in acute inflammation. Because of the key role of NO in each of these processes, abnormalities in vascular NO production are thought to contribute to the pathogenesis of certain vascular disorders such as those of atherosclerosis, diabetes, and hypertension. NO is synthesized in endothelial cells by oxidation of arginine catalyzed by the enzyme, endothelial nitric oxide synthase (eNOS). Obtaining a detailed understanding of the structure, function, and regulation of eNOS enzyme is thus clearly a worthy goal of scientific investigation. eNOS is regulated post-translationally by two primary mechanisms: protein-protein interactions and phosphorylation. Nothing is yet known about how these two different eNOS regulation processes are interrelated. What has been known for some time, however, is that there exists in eNOS an inhibitory phosphorylation site at serine 116 (S116). This site is phosphorylated in endothelial cells under basal conditions and after exposure to pro-inflammatory cytokines. Our preliminary data give rise to the hypothesis that proline-directed phosphorylation of eNOS at S116 promotes binding of the Pin1 prolyl isomerase and Pin1-induced conformational changes that suppress eNOS activity. Preliminary data also show that this process may make an important contribution to the disease of atherosclerosis. We therefore propose to test these hypotheses by a variety of different methods using purified proteins, cultured endothelial cells, intact blood vessel segments, and whole animals including animal disease models. These studies will provide a more complete understanding than currently exists of regulation of eNOS in endothelial cells, a topic of great interest to both basic scientists and clinicians. PUBLIC HEALTH RELEVANCE: Cardiovascular health critically depends on the maintenance of proper levels of nitric oxide (NO) in blood vessels to control blood pressure and to prevent the development of atherosclerosis. NO is produced in blood vessels by the endothelial nitric oxide synthase (eNOS). The primary objective of this research proposal is to obtain a more complete understanding of how eNOS is regulated by a combination of the two processes of phosphorylation and protein-protein interactions.